This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In addition to sperm, Drosophila males transfer dozens of proteins to females upon mating. Many of these proteins affect the physiology and behavior of females, and several are known to evolve rapidly between different Drosophila species. Previous computational work has predicted the set of proteins transferred in seminal fluid, but these predictions have been validated for only a handful of proteins. Here, we propose a mass spectrometry-based approach to comprehensively identify the set of male seminal fluid proteins. Drosophila melanogaster flies will be isotopically labeled by feeding on yeast labeled with a heavy isotope of nitrogen, 15-N. Labeled males will be mated to unlabeled females. Immediately following mating, we will dissect the female reproductive tracts and analyze the proteins therein by mass spectrometry. Male proteins will be distinguished from female proteins by the isotopic label. In this way, we aim to identify all of the proteins transferred in Drosophila seminal fluid. If this preliminary experiment is successful, we will proceed to test by the same method for variation in the levels and/or types of proteins transferred by different strains of D. melanogaster, by males facing different mating conditions, and by males of other Drosophila species. Our overall goal is to better understand the functions and evolution of proteins transferred during mating.